Identification and characterization of conjugated linolenic acid isomers by Ag+-HPLC and NMR

Interest in the biological activity of conjugated linolenic acids (CLnA) is growing. Technically, previous methods could not fully characterize and identify CLnA isomers. The present study is the first report on application of silver ion impregnated high-performance liquid chromatography (Ag+-HPLC) into separation of CLnA methyl ester (CLnAMe) mixture. Compared with the GC and reversed-phase HPLC techniques, Ag+-HPLC could resolve and separate CLnAMe isomers into three clusters of peaks namely ttt, ctt and ctc geometrical groups. Each positional isomer belonging to the same geometrical group could be further separated by Ag+-HPLC. Quantitative 13C NMR properties of CLnAMe mixture were subsequently studied. Olefinic signals in the CLnAMe mixture were assigned and used to quantify each isomer in combination with Ag+-HPLC. The present study provided a detailed analysis of a complex CLnA mixture and may have an important application in future studies dealing with synthesis, oxidative stability, and bioactivity of individual CLnA isomers.